EP0269017B1 - Association thérapeutique d'un épurateur de radicaux libres ou d'un inhibiteur métabolique et d'une protéine active biologiquement - Google Patents
Association thérapeutique d'un épurateur de radicaux libres ou d'un inhibiteur métabolique et d'une protéine active biologiquement Download PDFInfo
- Publication number
- EP0269017B1 EP0269017B1 EP87117149A EP87117149A EP0269017B1 EP 0269017 B1 EP0269017 B1 EP 0269017B1 EP 87117149 A EP87117149 A EP 87117149A EP 87117149 A EP87117149 A EP 87117149A EP 0269017 B1 EP0269017 B1 EP 0269017B1
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- Prior art keywords
- tnf
- lymphokine
- tumor
- cytotoxin
- biological
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- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This invention relates to a composition suitable for administration to mammalian hosts as a therapeutic formulation. More particularly, this invention relates to a combination therapy for free-radical bodily damage employing a lymphokine or cytotoxin such as tumor necrosis factor (TNF) and a biological modifier consisting of either one or more free radical scavengers that protect against damage caused by free-radical generation, or that selectively increase the susceptibility of a tumor to radical damage by depleting or reducing its radical scavenging capacity, or an inhibitor of one or both of the cyclooxygenase of lipoxygenase pathways of arachidonic acid metabolism.
- a lymphokine or cytotoxin such as tumor necrosis factor (TNF)
- TNF tumor necrosis factor
- a biological modifier consisting of either one or more free radical scavengers that protect against damage caused by free-radical generation, or that selectively increase the susceptibility of a tumor to radical damage by depleting or reducing its
- Lymphokines and cytotoxins are proteins secreted by T cells and/or macrophages upon activation by antigens or lectins.
- Interleukin-2 IL-2
- IL-2 Interleukin-2
- IL-2 a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan, D.A. et al., Science (1976) 193 :1007-1008.
- T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes, it is now recognized that in addition to its growth factor properties it modulates a variety of functions of immune system cells in vitro and in vivo and has been renamed interleukin-2 (IL-2).
- IL-2 is one of several lymphocyte-produced, messenger-regulatory molecules which mediate immunocyte interactions and functions.
- Tumor necrosis factor was first described by Carswell et al., Proc. Natl. Acad. Sci. USA (1975) 72 :3666-3670 as an endotoxin-induced serum factor which causes necrosis of chemically transformed tumor cells when growing in mice.
- Human TNF is known to be cytotoxic to neoplastic cells, and has been produced in recombinant form. See Pennica et al., Nature (London) (1984) 312 :724-729 and Shirai et al., Nature (London) (1985) 313 :003-806, Wang et al., Science (1985) 228 :149-154.
- Interferons constitute a group of naturally occurring proteins which are known to exhibit anti-viral, anti-tumor and immunoregulatory behavior. Two types of IFN have been identified based on differences in their observed biological properties and molecular structures: Type I and Type II.
- Beta-interferon IFN-ß
- IFN-ß is a Type I IFN which can be induced in fibroblasts by viral challenge and contains about 165 amino acids.
- IFN- ⁇ is also a Type I IFN inducible in leukocytes, and IFN- ⁇ is a Type II IFN which is induced in lymphocytes in response to specific mitogenic stimuli and contains 146 amino acids.
- Combination chemotherapy using two or more anti-cancer drugs to treat malignant tumors in humans is currently in use in research and in the clinic.
- the anti-cancer drugs may be antimetabolites, alkylating agents, antibiotics, general poisons, etc.
- Combinations of drugs are administered in an attempt to obtain a synergistic cytotoxic effect on most cancers, e.g., carcinomas, melanomas, lymphomas and sarcomas, and to reduce or eliminate emergence of drug-resistant cells and to reduce side effects to each drug.
- IL-2 may be used with IFN- ⁇ to treat tumor-bearing hosts with synergistic results [EP-A-0 149 551, published July 24, 1985 (Genentech) and DE-A-3411184, published October 31, 1985 (Deut. Rotes Kreuz )] or with augmentation of natural killer activity [Svedersky et al ., J. Immunol. (1984), 133 :714-718 and Shalaby et al., J. Interferon Res. (1985), 5 :571-581].
- synergistic results [EP-A-0 149 551, published July 24, 1985 (Genentech) and DE-A-3411184, published October 31, 1985 (Deut. Rotesdorf )] or with augmentation of natural killer activity [Svedersky et al ., J. Immunol. (1984), 133 :714-718 and Shalaby et al., J. Interferon Res. (1985), 5 :571-581].
- TNF which is produced primarily by macrophages, has shown an apparent selectivity for many tumor cells, but not normal cells, in its cytotoxic or cytostatic activities. See, e.g., Carswell et al., supra , Wang et al., supra , Ruff and Gifford in Lymphokines, Volume 2 , ed. Pick, E. (Academic Press, Inc., NY, NY, 1981), pp.
- TNF r cells such as human diploid fibroblasts
- TNF r cells have sufficient numbers of high affinity receptors, internalize TNF, and degrade it in an apparently similar fashion as TNF s cells do.
- Interleukin-1 alone has a protective effect in a model of free radical dependent tissue injury, Neta et al., J. Immunol. (1986) 136 :2483-2485.
- TNF- ⁇ and IFN- ⁇ induce neutrophils from normal and chronic granulomatous-disease patients to release superoxide, Palladino et al., Clin. Res. (1986) 34 :502 and Palladino et al., Ped. Res. (1986) 20 :302.
- oxygen radicals can initiate longer-lived chain reactions of lipid peroxidation that can be propagated from cell to cell. These peroxidation products are capable of damaging cellular DNA, RNA, protein, and cellular phospholipids. See, e.g., Slater, supra , Bus, supra , Moody and Hassan, Proc. Natl. Acad. Sci.
- the protective cellular mechanisms against this kind of damage include anti-oxidants and radical scavengers in both the lipid (e.g., ⁇ -tocopherol, ⁇ -carotene) and aqueous (e.g., glutathione and ascorbic acid) phases of cells, as well as enzymes such as superoxide dismutase and catalase.
- lipid e.g., ⁇ -tocopherol, ⁇ -carotene
- aqueous e.g., glutathione and ascorbic acid
- enzymes such as superoxide dismutase and catalase.
- Glutathione (GSH) and related cellular sulfhydryl compounds represent one of the major mechanisms of detoxification of electrophilic metabolites of xenobiotics and oxygen/lipid radical species, Meister and Anderson, supra . Inhibition of free radicals is postulated as the way in which certain radioprotectors, such as the free radical scavengers cysteine and GSH, operate. GSH becomes oxidized to contain a dithio group as well as to protein-mixed disulfides, when cells are exposed to oxygen-generating compounds or other oxidative stresses. See Adams et al., J. Pharmacol. Exp. Ther. (1983) 227 :749-754.
- oxidized GSH is one important indicator of either the type of damage to which a cell has been exposed or of its ability to protect itself from oxidative damage.
- Buthionine sulphoximine has been shown to be an inhibitor of GSH biosynthesis. See Minchinton et al., Int. J. Radiation Oncology Biol. Phys. (1984) 10 :1261-1264.
- MCCT monocyte cell line cytotoxin
- Andrews et al., Cancer Res. (Dec. 1985) 45 :6250-6253 discloses the differential potentiation of alkylating and platinating agent cytotoxicity in human ovarian carcinoma cells by glutathione depletion.
- Oxols et al., Dev. Oncol. (1986) 47 :277-293 discloses the effect of BSO on the efficacy of antitumor drugs.
- Crook et al., Cancer Res. (1986) 46 :5035-5038 discloses that BSO enhances the cytotoxicity of cyclophosphamide.
- Hodgkiss et al., Biochem. Pharmacol. (1985) 34:2175-2178 discloses use of BSO to enhance the cytotoxicity of nitroaromatic compounds.
- Somfai-Relle et al., Biochem. Pharmacol. (1984) 33 :485-490 discloses that BSO sensitizes murine tumor cells to L-phenylalanine mustard.
- EP-A-0 177 342 discloses pharmaceutically acceptable formulations designed for the purpose of administering certain amphophilic proteins combined with lipophilic vehicles and GI tract adsorption enhancing agents in oral dosage form. Said formulations do not comprise biological modifiers as free radical scavengers or metabolic inhibitors.
- the present invention is based on the finding that the therapeutic index of a lymphokine or cytotoxin can be enhanced in in vitro and in vivo systems by concomitantly or separately treating the host with a free radical scavenger such as a radiation protector or a metabolic inhibitor in amounts that increase the efficacy and/or decrease the toxicity of the lymphokine or cytotoxin.
- a free radical scavenger such as a radiation protector or a metabolic inhibitor
- the present invention is directed to a composition suitable for administration to mammalian hosts for therapeutic or prophylactic treatment of biological damage to mammalian hosts caused by free radical production
- composition comprises a mixture, in pharmacologically effective amounts, of at least one lymphokine or cytotoxin from a mammalian species and at least one biological modifier selected from a free radical scavenger or a metabolic inhibitor.
- the lymphokine or cytotoxin is tumor necrosis factor or interleukin-2 and the free radical scavenger or metabolic inhibitor is uric acid, buthionine sulphoximine, vitamin C, indomethacin, ibuprofen, N-acetyl cysteine, or aspirin.
- lymphokine or cytotoxin such as TNF
- free-radical scavenging capacity activation of the arachidonic acid cascade may be involved in the mechanism of action of the lymphokine or cytotoxin, which can produce lipid peroxidation and other associated radical species, as well as the biologically active metabolites of the lipoxygenase and cyclooxygenase pathways.
- lymphokine refers to low molecular weight proteins that are secreted by T cells and/or macrophages when antigens or lectins stimulate T cell or macrophage growth or activation.
- cytotoxin refers to any protein that activates effector cells that kill foreign agents such as pathogens in the cell.
- lymphokines and cytotoxins include, but are not limited to, interferons [e.g., interferon-alpha, (IFN- ⁇ ), interferon-beta, (IFN- ⁇ ), and interferon-gamma, (IFN- ⁇ )], interleukins [e.g., interleukin-1 (IL-1), Interleukin-2 (IL-2), interleukin-3 (IL-3), and interleukin-4 (IL-4)], tumor necrosis factor-alpha (TNF- ⁇ ), tumor necrosis factor-beta (TNF- ⁇ ) (also called lymphotoxin), a colony stimulating factor (eg.
- interferons e.g., interferon-alpha, (IFN- ⁇ ), interferon-beta, (IFN- ⁇ ), and interferon-gamma, (IFN- ⁇ )
- interleukins e.g., interleukin-1 (IL-1), Interleukin-2 (IL-2), interleukin-3 (
- the lymphokine or cytotoxin is an interleukin (more preferably IL-2), an interferon (more preferably IFN- ⁇ ), TNF- ⁇ or - ⁇ , or a colony stimulating factor (more preferably CSF-1). The most preferred herein is TNF- ⁇ .
- the term "recombinant” refers to lymphokines or cytotoxins produced by recombinant DNA techniques wherein generally the gene coding for the lymphokine or cytotoxin is cloned by known recombinant DNA technology.
- a suitable DNA vector such as a bacterial plasmid, preferably an E. coli plasmid, to obtain a recombinant plasmid, and the plasmid is used to transform a suitable host.
- the gene is expressed in the host to produce the recombinant protein.
- suitable recombinant plasmids for this purpose include pBR322, pCR1, pMB9 and pSC1.
- the transformed host may be eukaryotic or prokaryotic, preferably, a prokaryotic host.
- the term “pharmaceutically acceptable” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the hosts to which it is administered.
- the term "prophylactic or therapeutic" treatment refers to administration to the host of the lymphokine(s) or cytotoxin(s) and biological modifier(s) either before or after onset of the biological damage to the host. If the lymphokine(s) or cytotoxin(s) and biological modifier(s) are administered prior to exposure to the agent causing the biological damage, the treatment is prophylactic (ie., it protects the host against the damage), whereas if it is administered after exposure to the agent causing the damage, the treatment is therapeutic (ie., it alleviates the existing damage).
- the scheduling and dosing will depend, e.g., on the type of host, disease, lymphokine or cytotoxin, and biological modifier. If the biological damage is caused by infection, the doses are preferably administered from 18 hours before infection for prophylactic treatment and in early phase of infection for therapeutic treatment, up to 18 hours after infection in later phase of infection for therapeutic treatment.
- Prophylactic treatment of cancer refers to administration after the patient has been treated for cancer, to prevent reoccurrence of the cancer.
- the term "biological damage to the host caused by free radical generation” refers to any cellular, tissue or other damage to body parts or functions sustained by the host as a result of free radicals being produced in the body of the host.
- the free radicals may cause directly mobilization of the arachidonic acid metabolic pathways or may cause lipid peroxidation that mobilizes arachidonic acid. These radicals may be produced as a mechanism for killing cells. Examples by which such damage may be caused include hyperthermia, which may occur during cancer treatment as when the temperature of the tumor is increased via local or general microwave irradiation, damage caused by chemotherapeutic agents (chemotherapy), radiation therapy, or high oxygen tension that produce radicals to kill cells, and infection. Also, treated tumor cells may help propagate radical damage. An example of high oxygen tension is the condition that occurs when premature babies are exposed to high pressure oxygen, resulting in retinal and lung disease. Other conditions that represent damage caused by free radical generation may also be envisioned and fall within this definition.
- cancer refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer and gastrointestinal or stomach cancer.
- the cancer is colon cancer, melanoma, renal cell cancer, sarcoma, lung cancer, adenocarcinoma, or breast cancer.
- infection refers to any kind of pathogenic disease, including those caused by bacteria, fungi, viruses, protozoa or parasites.
- bacterial infections include P. aeruginosa , E. coli , tetanus, Mycobacterium species, Streptococcal strains, diphtheria and Salmonella.
- fungal infections include cryptococcosis, histoplasmosis, and other infections due to Candida species.
- viral infections include Hepatitis A, recurrent Herpes Simplex, AIDS, Herpes Zoster, influenza, and rhinoviruses.
- the infection is bacterial, more preferably Gram-negative infection, and most preferably P. aeruginosa and E. coli infection.
- the term "biological modifier” refers to one of two compounds: a free radical scavenger or a metabolic inhibitor.
- free radical scavenger refers to any compound or substance that protects a mammalian host against biological damage caused by free radical generation. This definition includes those agents that act through direct radical scavenging as well as those that act by altering the radical scavenging ability of the host and/or tumor. Either mechanism will affect the response of the host to the lymphokine or cytotoxin.
- Such free radical scavengers may be radiation protectors, and include such compounds as, for example, uric acid, buthionine sulfoximine, diethyl maleate, vitamin E, vitamin C, cysteine such as N-acetyl cysteine, or glutathione, metronidazole, and a retinoid such as, e.g., vitamin A. While diethyl maleate may increase the toxicity of the lymphokine or cytotoxin, it also reduces glutathione preferentially in tumor versus host and therefore should lead to a higher therapeutic index. Any combination of these modifiers may be employed.
- the free radical scavenger employed herein for humans is buthionine sulphoximine, vitamin C, vitamin E, or N-acetyl cysteine [the latter having the tradename Mucomyst (Mead Johnson)]. See the 1987 Physicians' Desk reference, ed. 41, Barnhart, pub., Oradell, N.J: Medical Economics Company, Inc. Uric acid causes gout in humans so it may be tolerated in lesser amounts; it occurs naturally in human plasma at about 300 ⁇ M; it is expected that about 600-1500 ⁇ M might be tolerated in humans.
- the second type of biological modifier refers to a compound or substance that blocks or inhibits the cyclooxygenase and/or lipoxygenase metabolic pathways, of the arachidonic acid cascade, wherein phospholipids are converted to arachidonic acid, by phospholipase A2 or C, and the arachidonic acid may proceed by either metabolic pathway.
- a metabolic inhibitor refers to a compound or substance that blocks or inhibits the cyclooxygenase and/or lipoxygenase metabolic pathways, of the arachidonic acid cascade, wherein phospholipids are converted to arachidonic acid, by phospholipase A2 or C, and the arachidonic acid may proceed by either metabolic pathway.
- Such blockage or inhibition may be of an enzyme that catalyzes one or both pathways, of a cell type that contains the enzyme, or of one or more of the natural products of the pathways.
- metabolic inhibitors examples include aspirin, indomethacin, ibuprofen, nordihydroguaiaretic acid (4,4'-[2,3-dimethyl-1,4-butanediyl]-bis[1,2-benzenediol]) (NDGA), cis-8,11,14-eicosatrien-5-ynoic acid (ETYA), and synthetic (as opposed to natural) prostaglandins and/or leukotrienes that block the effects of the natural metabolic products at the production level rather than at the enzyme level.
- indomethacin, ibuprofen, and ETYA block the cyclooxygenase pathway, thereby inhibiting the production of natural prostaglandins, thromboxanes and prostacyclins. At higher concentrations, indomethacin also blocks phospholipase A2. NDGA and ETYA block the lipoxygenase pathway, thereby inhibiting the production of natural hydroperoxy-eicosatetraenoic acid, leukotrienes, and hydroxy- and dihydroxy-eicosatetraenoic acids.
- the preferred metabolic inhibitors herein for use with TNF are those selected from aspirin, indomethacin, and ibuprofen. Indomethacin is a non-steroidal antirheumatic agent with local antiinflammatory activity. Ibuprofen is a substitute for aspirin. Both drugs are referred to in the 1987 Physician's Desk Reference , supra .
- the term "pharmacologically effective amounts" as applied to the lymphokine or cytotoxin and the biological modifier refers to the amount of each component in the mixture or administered to the host that results in an increase in the therapeutic index of the host.
- the "therapeutic index” can be defined for purposes herein in terms of efficacy (extent of tumor or infection reduction or other cure) and in terms of toxicity to the host.
- the therapeutic index has increased.
- the ratio of mean body weights indicates the extent of toxicity, with a value of 1 indicating no toxicity.
- the extent of efficacy achieved may be measured by the ratio of mean tumor volume at the end of the evaluation period for efficacy response to mean tumor volume at the start of treatment.
- a reduction in the ratio of at least 50% of treated over excipient control indicates increased efficacy.
- the most preferred doses, schedules, and types of biological modifers are those that achieve a mean tumor volume ratio of between 0 and 5, with a value of 0 being optimum and indicating a cure.
- the efficacy increases at least 50% upon treatment with the lymphokine/cytotoxin and biological modifiers and the toxicity is acceptable, i.e., no more than fever, chills, and/or general malaise, the therapeutic index has increased.
- the extent of efficacy is generally ascertained in the clinic by measuring the perpendicular diameters of the products of all measured disease. A partial response occurs when the tumor shrinks by at least 50% in the sum of the products of the perpendicular diameters of all measured disease.
- a tumor having perpendicular diameters of 10 and 10 shrinks to perpendicular diameters of 8 and 8, the tumor has only shrunk from 100 to 64, which is not a 50% reduction and is not a partial response. If the tumor of 10 and 10 shrinks to 7 and 7, however, this is a partial response because it has shrunk from 100 to 49, more than 50%.
- lymphokine(s), cytotoxin(s) and biological modifier(s) may be combined in vitro before administration or separately administered to the host, in either order or concurrently or simultaneously, with any administration of lymphokine/ cytotoxin generally taking place up to 24 hours after the administration of biological modifier, and with any administration of biological modifier taking place up to about one hour after the administration of lymphokine/cytotoxin.
- the biological modifier is added prior to adding or concurrently with the lymphokine or cytotoxin.
- the administration(s) may take place by any suitable technique, including oral, subcutaneous and parenteral administration, preferably parenteral or oral.
- parenteral administration include intravenous, intraarterial, intramuscular, and intraperitoneal, with intraperitoneal and intravenous being preferred.
- the dose and dosage regimen will depend mainly on whether the lymphokine(s) or cytotoxin(s) and biological modifier(s) are being administered for therapeutic or prophylactic purposes, separately or as a mixture, the type of biological damage and host, the history of the host, the type of lymphokine or cytotoxin, and the type of biological modifier employed.
- the amount must be effective to achieve an enhanced therapeutic index as defined above. It is noted that humans are treated longer than the mice and rats exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness.
- the doses may be single doses or multiple doses over a period of several days, but single doses are preferred.
- a protection level of at least 50% means that at least 50% of the treated hosts exhibit improvement against the infection, including but not limited to improved survival rate, more rapid recovery, or Improvement or elimination of symptoms.
- LAK cells are lymphoid cells that can lyse fresh, noncultured, natural-killer-cell-resistant tumor cells but not normal cells.
- the doses may be single doses or multiple doses. If multiple doses are employed, as preferred, the frequency of administration will depend, for example, on the type of host and type of cancer, dosage amounts, etc. For some types of cancers or cancer lines, daily administration may be effective, whereas for others, administration every other day or every third day may be effective, but daily administration ineffective. The practitioner will be able to ascertain upon routine experimentation which route of administration and frequency of administration are most effective in any particular case.
- the dosage amounts for cancer which appear to be most effective herein are those that result in regression in size of the tumor or complete disappearance or non-reappearance of the tumor, and are not toxic or are acceptably toxic to the host patient. Generally, such conditions as fever, chills and general malaise are considered acceptable.
- the dose of biological modifier cannot be so large as to inhibit the anti-tumor activity of the lymphokine or cytotoxin. These optimum dose levels will depend on many factors, for example, on the type of host, cancer, route, schedule and sequence of administration, existing tumor burden, the type of lymphokine or cytotoxin and biological modifier, and the definition of toxicity.
- Toxicity may be defined by the extent and type of side effects in human hosts, with fever, chills and general malaise considered acceptable toxicity for the study herein, or by the amount of body weight loss or by death in non-human hosts after a certain period of time, as defined above for the therapeutic index.
- TNF- ⁇ is employed as the lymphokine or cytotoxin
- the dosage level thereof in humans is generally at least 0.24 ⁇ g/kg patient weight, and in mice is at least 25 ⁇ g/kg.
- the amount of TNF- ⁇ administered to humans is the approximate or exact number that is used for mice, but the units are ⁇ g/m2 rather than ⁇ g/kg.
- TNF- ⁇ is administered to humans in an amount of about 25 to 100 ⁇ g/m2 when buthionine sulphoximine is administered in a minimum effective radical scavenging concentration before and/or during the course of administration of the TNF- ⁇ . Smith et al., Proceedings of AACR , 28 (March 1987), p.
- TNF- ⁇ is administered to humans in an amount of 125 ⁇ g/m2 when vitamin C is administered in a minimum effective radical scavenging concentration prior to administration of the TNF- ⁇ .
- TNF- ⁇ is administered to humans in an amount of 25-100 ⁇ g per m2 when aspirin is administered in an amount of about 15-30 mg/kg of patient weight prior (e.g., 1-4 hours) to administration of TNF- ⁇ .
- TNF- ⁇ is administered to humans in an amount of about 50-200 ⁇ g/m2 when indomethacin is administered in an amount of about 25-50 mg prior to administration of the TNF- ⁇ .
- TNF- ⁇ is administered to humans in an amount of about 150-250 ug/m2 host when ibuprofen is administered in an amount of about 400 to 600 mg, when ibuprofen is administered prior to administration of TNF- ⁇ .
- TNF- ⁇ is administered to humans in an amount of about 200-400 ⁇ g/m2 when N-acetyl cysteine is administered in an amount equivalent to 250-1000 mg/kg of rat weight prior to the administration of TNF- ⁇ .
- the practitioner will be able to determine optimum dosage levels and scheduling when the host, lymphokine/cytotoxin and biological modifier are varied.
- the dosage level thereof in humans is generally at least about 3 x 106 units/m2/day and in mice is at least about 5-10 mg/kg.
- the IL-2 is administered to humans in an amount of at least 3 x 106 units/m2/day when vitamin C, vitamin E, aspirin, N-acetyl cysteine, ibuprofen or indomethacin is administered prior to administration of the IL-2.
- the dosage level of CSF-1 in humans has not yet been determined, but in mice it may generally be about 50 mg/kg (at 100-150 mg/kg the mice being to die).
- the typical dosage level of interferon (especially INF- ⁇ ) in humans ranges from about 100 units to one billion units/m2.
- IFN- ⁇ is administered to humans in an amount of at least 1000 units/m2 when vitamin C, vitamin E, aspirin, N-acetyl cysteine, ibuprofen, or indomethacin is administered prior to administration of the IFN- ⁇ .
- the lymphokine(s) will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion), preferably in a pharmaceutically acceptable carrier medium which is inherently non-toxic and non-therapeutic or non-prophylactic.
- a pharmaceutically acceptable carrier medium which is inherently non-toxic and non-therapeutic or non-prophylactic.
- examples of such vehicles include water, saline, Ringer's solution, dextrose solution, mannitol, and normal serum albumin.
- Non-aqueous vehicles such as fixed oils, propylene glycol and ethyl oleate may also be used.
- the carrier medium may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
- the lymphokine(s)/cytotoxins and biological modifiers will typically be formulated in such carriers at a concentration of about 0.1 mg/ml to 100 mg/ml of each, preferably 0.2 to 1 mg/ml of
- the lymphokine is IL-2
- it may be made into a, sterile, stable lyophilized formulation in which the purified IL-2 is admixed with a water-soluble carrier such as mannitol, which provides bulk, and a sufficient amount of sodium dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water.
- a water-soluble carrier such as mannitol, which provides bulk, and a sufficient amount of sodium dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water.
- the formulation is suitable for reconstitution in aqueous injections for parenteral administration and it is stable and well-tolerated in human patients.
- the formulation method is more completely described in U.S. Patent No. 4,604,377.
- the IL-2 may be refolded using guanidine to obtain a more soluble product. Guanidine may be used to solubilize the IL-2 particle
- the guanidine may be used after the HPLC step, in a process described below.
- the rIL-2 is separated from the bulk of the cellular components of the transformed microorganism hosts containing the rIL-2, the rIL-2 is solubilized in a reduced form, oxidized, purified to clinically acceptable purity and endotoxin levels, and denatured by placing the rIL-2 in a solution of a chaotropic agent. Thereafter, the solids are removed from the solution and the rIL-2 is renatured from the solution.
- the solution of a chaotropic agent is a 4 to 8 M aqueous guanidine hydrochloride solution.
- the IL-2 may be administered in an adoptive immunotherapy method, together with isolated, lymphokine-activated lymphocytes, in a pharmaceutically acceptable carrier, where the lymphocytes have antitumor activity when administered with the IL-2 to humans suffering from the tumor.
- This method is described more fully In U.S. Patent No. 4,690,915 issued September 1, 1987, and by S. Rosenberg et al., New England Journal of Medicine (1985), 313 :1485-1492.
- TIL tumor-infiltrating lymphocytes expanded in IL-2 may be adoptively transferred for the therapeutic treatment, particularly in combination with cyclophosphamide.
- TIL approach may also be used herein.
- the recombinant lymphokine employed herein may be any lymphokine, obtained from tissue cultures or by recombinant techniques, and from any mammalian source such as, e.g., mouse, rat, rabbit, primate, pig, and human.
- the lymphokine is derived from a human source and more preferably is a human recombinant lymphokine.
- the lymphokine is recombinant human IL-2 or TNF alone or in combination with recombinant TNF or IL-2, respectively.
- the recombinant IL-2 may be obtained as described by Taniguchi et al., Nature , 302 :305-310 (1983) and Devos, Nucleic Acids Research , 11 :4307-4323 (1983) by cloning the native human IL-2 gene and expressing it in transformed microorganisms. It may also be an IL-2 mutein as described in U.S. Patent No.
- the IL-2 is an unglycosylated protein which is produced by a microorganism which has been transformed with the human cDNA sequence or a modified human cDNA sequence of IL-2 which encodes a protein with an amino acid sequence at least substantially identical to the amino acid sequence of native human IL-2, including the disulfide bond of the cysteines at positions 58 and 105, and has biological activity which is common to native human IL-2.
- Substantial identity of amino acid sequences means the sequences are identical or differ by one or more amino acid alterations (deletions, additions, substitutions) which do not cause an adverse functional dissimilarity between the synthetic protein and native human IL-2.
- IL-2 proteins with such properties include those described by Taniguchi et al., Nature (1983), 302 :305-310; Devos, Nucleic Acids Research (1983), 11 :4307-4323; and by EP-A-0 091 539 and 0 088 195; in U.S. Patent 4,518,584, supra , and in EP-A-0 200 280, supra .
- the IL-2 is the desala1-IL-2 ser125 mutein in which the initial terminal alanine is deleted and the cysteine at position 125 is replaced by a serine residue.
- the IL-2 may be produced and purified to clinical purity by the method described and claimed in U.S. Patent No. 4,569,790, issued February 11, 1986.
- the IL-2 may be recovered from refractile bodies using sucrose as described in EP-A-0 206 828, published December 30, 1986.
- the IL-2 may also be modified by derivatization with polyethylene glycol or a polyoxyethylated polyol, as described in PCT 87/00056, published January 15, 1987.
- the human TNF- ⁇ herein may be obtained in recombinant form as described by Pennica et al., Nature (1984) 312 :724-729; Yamada et al., J. Biotechnology , (1985) 3 :141-153; Wang et al., Science , (1985) 228 :149-154; Shirai et al., Nature (London), (1985) 313 :803-806; EP-A-0 155 549, published September 29, 1985; EP-A-0 158 286, published October 16, 1985; EP-A-0 168 214, published January 15, 1986; and PCT US85/01921, published April 24, 1986.
- Recombinant rabbit TNF- ⁇ may be obtained as described in EP-A-0 146 026, published June 26, 1985 and EP-A-0 148 311, published July 17, 1985.
- the TNF- ⁇ herein is a human TNF mutein wherein one or more of the first eight amino acid residues, preferably either the first four or the first eight, have been deleted, using the procedure described in U.S. Patent No. 4,677,064, supra , or the TNF- ⁇ is a cysteine-depleted mutein described in U.S. Patent No. 4,677,063 issued June 30, 1987, supra , and in U.S. Patent No. 4,518,584, supra .
- the TNF may be purified by the method described in EP-A-0 220 966, published May 6, 1987.
- TNF- ⁇ The precise chemical structure of the TNF- ⁇ herein will depend on a number of factors. As ionizable amino and carboxyl groups are present in the molecule, a particular form of TNF- ⁇ may be obtained as an acidic or basic salt, or in neutral form. All such preparations which retain their bioactivity when placed in suitable environmental conditions are included in the definition of the TNF- ⁇ herein. Further, the primary amino acid sequence of the TNF- ⁇ may be augmented by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate or acetyl groups, more commonly by conjugation with saccharides. Certain aspects of such augmentation are accomplished through posttranslational processing systems of the producing host; other such modifications may be introduced in vitro .
- TNF- ⁇ modifications are Included in the definition of TNF- ⁇ herein so long as the bioactivity of the TNF- ⁇ is not destroyed. It is expected, of course, that such modifications may quantitatively or qualitatively affect the bioactivity by either enhancing or diminishing the activity of the TNF- ⁇ in the various assays.
- the TNF- ⁇ may be reacted with a homopolymer or copolymer of polyethylene glycol or a polyoxyethylated polyol, provided that the polymer is soluble in water at room temperature.
- the polymer is reacted first with a coupling agent having terminal groups reactive with both the free amino or thiol groups of the protein and the hydroxyl group of the polymer.
- Examples of such coupling agents include hydroxynitrobenzene sulfonic ester, cyanuric acid chloride and N-hydroxysuccinimide.
- the TNF- ⁇ is then formulated directly with the water-soluble carrier and buffer as described above, and the formulation may be lyophilized and the lyophilized mixture reconstituted as described above.
- Recombinant IFN- ⁇ may be obtained as described by Gray et al., Nature , 295 :503 (1982).
- mice Female Balb/c mice (Charles River Breeding Laboratories, Inc., Wilmington, MA) that were all 6-8 weeks old were employed in the in vivo tests. Animals were weight matched at 20 ⁇ 3 g, randomized at five per cage, and ear-notched. All animals were held in quarantine observation for seven days after arrival, maintained in microisolator cages (Lab Products, Inc.), and fed standard laboratory diets and drinking water ad lib.
- TNF- ⁇ having the first eight amino acids deleted from the N-terminus was prepared as described In U.S. Patent No. 4,677,064 Issued June 30, 1987 and Wang et al., Science (1985) 228 :149-153. Briefly, TNF was induced from HL-60 cells and purified and sequenced. Then an intronless sequence encoding human TNF was prepared by producing enriched mRNA, constructing a cDNA library, selecting a probe and probing the library to recover the sequence. Then an ATG start codon was introduced immediately preceding the GTC sequence encoding N-terminal valine of the mature protein by site-directed mutagenesis.
- Clones were selected and strands ligated into expression vectors to obtain prokaryotic expression of the mutein.
- the mutein was then purified by column purification using any standard purification technique and recovered in the purification buffer.
- the muteln was produced as a lyophilized powder in sterile vials, reconstituted and suspended using sterile phosphate buffered saline within four days prior to use, and stored, if at all, at 4°C.
- the TNF contained less than 0.001 to 0.006 ng endotoxin/mg protein depending on production lot.
- the free radical scavenger employed herein was commercially available uric acid. (Sigma).
- the target cells employed were a methylcholanthrene-induced murine fibrosarcoma (Meth-A) (Balb/c) obtained as an ascites-passed tumor from Dr. Lloyd Old, Memorial Sloan-Kettering Cancer Center, New York, New York, frozen as stock, and passed at least twice in ascites prior to use. These cells were implanted subcutaneously in the suprascapular area of the mouse host.
- Method-A methylcholanthrene-induced murine fibrosarcoma
- Table I indicates the results obtained when the TNF mutein alone, uric acid alone, and various combination injections of the TNF mutein and uric acid were administered intravenously to five mice per group (2-3 repeats) beginning seven days after tumor implantation and every third day three times, with final measurements made on the 14th day.
- the excipient control was injected with PBS.
- the uric acid dose was slightly below the maximum concentration attainable without decreasing animal weight, determined in a preliminary toxicity study, and was given immediately prior to the TNF inoculation.
- the TNF doses were chosen to cover the ranges of 50, 100, 150, 200 and 250 ⁇ g/kg of mice weight (the mice weighing approximately 20 g each).
- the uric acid doses were 25 mg/kg of mouse weight whenever uric acid was administered.
- b ⁇ TW change in tumor volumes as measured by the ratio of mean tumor volume (in mm3) at 14 days after treatment to mean tumor volume (in mm3) at the start of treatment.
- mice resulted in a decrease in toxicity over both PBS control and TNF alone as measured by host death, and an increase in efficacy as measured by comparing the ⁇ TW of the PBS control and the ⁇ TW of the combinations. Therefore, the therapeutic index as defined herein has been enhanced.
- Uric acid has been postulated by Ames et al., supra , as a major protective agent against damage by reactive oxygen species and lipid peroxidation present in primates, but not rodents.
- the results of these experiments indicate that reactive oxygen radicals and/or lipid peroxide-like products play a role in the mechanism of action of TNF.
- the practitioner can predict that these results likely would apply to humans based on the expected correlation between the TNF dose-related anti-tumor effect in animals and humans.
- the preclinical response of TNF alone correlated with a clinical response of TNF to colon cancer.
- mice Female Balb/c mice were implanted subcutaneously with Meth-A tumor cells as described in Example 1.
- Table II indicates the results obtained when either vitamin C or the TNF mutein of Example 1 alone, or various combinations of vitamin C immediately followed by the TNF mutein were administered intravenously to groups of 5 mice beginning seven days after tumor implantation and continuing every third day for three injections, with measurements taken on the fourteenth day following the initiation of treatment.
- b ⁇ TW change in tumor volumes as measured by the ratio of mean tumor volume (in mm3) at 14 days after treatment to mean tumor volume (in mm3) at the start of treatment; % control is the normalized value relative to the control groups.
- Vitamin C functions as an aqueous compartment radical scavenger, as well as in collagen stability.
- mice Female Balb/c mice were implanted subcutaneously with Meth-A tumor cells as described in Example 1. Treatment was initiated seven days after the implantation.
- BSO or the TNF mutein of Example 1 alone, or BSO together with the TNF mutein were administered.
- BSO was administered intraperitonally and TNF intravenously.
- the BSO was started 24 hours prior to TNF administration twice daily (6-8 hours between doses) for ten days and TNF was given with the second BSO dose every third day for three times.
- BSO excipient was used as a volume control in the TNF alone groups.
- PBS was used as a control.
- the protocol is illustrated below:
- Table III illustrates the results of this study, with the headings defined in the footnotes to Table II. The numbers in parentheses indicate the results from a repeat of the experiment.
- TABLE III Inhibition of Glutathione Synthesis by Buthionine Sulphoximine Increased the Sensitivity of Meth-A tumor cells to TNF In Vivo a Treatment Group ⁇ BW b Deaths ⁇ TW c Cures d
- Table IV illustrates the sensitivity of various human cell lines to TNF and the relationship of TNF sensitivity to glutathione (GSH) content In the cells.
- BSO is used to reduce preferentially the GSH level of Meth-A as compared to the host, thereby depleting the radical scavenging capacity of the tumor. All cell lines mentioned below are available from the American Type Culture Collection (ATCC) in Rockville, MD.
- mice were treated daily for 14 days i.p., at 250 ⁇ g/kg, one day following subcutaneous tumor inoculation.
- PAN-02 and HT29 were treated every third day, three times, i.v.,, at 150 or 100 ⁇ g/kg, respectively, starting 7 days after tumor inoculation.
- mice Female Balb/c mice were treated by subcutaneous implantation of Meth-A tumor cells as described in Example 1. Five mice per group were employed. Treatment was initiated seven days after the implantation.
- a protocol was employed wherein aspirin or the TNF mutein of Example 1 alone, or aspirin together with the TNF mutein , were administered intravenously.
- aspirin was administered daily for five days and each dose was followed 1-4 hours later by TNF.
- PBS was used as a control.
- mice Female Balb/c mice were treated by subcutaneous implantation of Meth-A tumor cells as described in Example 1.
- Table VI Indicates the results obtained when NDGA (from Sigma Chemical Co.), aspirin, the combination of NDGA and aspirin, the TNF mutein of Example 1 alone, and various combinations of NDGA and aspirin followed by the TNF mutein were administered to groups of 5 mice beginning seven days after tumor implantation and continuing every third day three times. Details of the protocols are in the footnotes of Table VI. PBS was used as a control. Results were evaluated 14 days after initiation of treatment. The headings are defined in the footnotes of Table I.
- b 30 mg/kg iv. one hour prior to each TNF administration.
- c 312 ⁇ g/20 g mouse (15.6 mg/kg) in propylene glycol i.p. 5 minutes prior to each TNF administration.
- d i.v. every third day 3 times.
- e i.p. every third day 3 times.
- f aspirin i.v. every third day 3 times; NDGA i.p. one hour after each aspirin administration
- NDGA is known to inhibit the 5'-lipoxygenase pathway, which would be expected to inhibit leukotriene synthesis and, thereby, the toxicities associated with their production in the host.
- NDGA may function in a radical scavenging role in some cases, which role would be expected also to reduce the toxicity of TNF. The exact mechanisms at work under the present conditions are not entirely known at this time, but evidence has been presented to suggest that either effect or both effects would be expected to reduce TNF toxicity.
- mice Female Balb/c mice were treated by subcutaneous implantation of Meth-A tumor cells as described in Example 1.
- Table VII indicates the results obtained when aspirin, indomethacin, ibuprofen, the TNF mutein of Example 1 alone, and either aspirin, indomethacin or ibuprofen followed in two hours by the TNF mutein were administered to groups of 10 mice and of five mice beginning seven days after tumor implantation. Details of the protocols are referenced in the footnotes of Table VII. Results were evaluated 14 days after initiation of treatment for TGI, and at least 21 days, usually 28 days, for cure data. TGI is tumor growth Inhibition, calculated as the % ratio of the weight of the treated tumor at day 14 to the weight of the tumor at day 14 of the controls, subtracted from 100. (For example, if treated volume is 30 and control volume is 1200, the ratio is 2.5%, and the % TGI is 97.5.) Cures are the same as defined in footnote c of Table I.
- mice of TNF for 3 mg/kg indomethacin show the best dosage for mice of TNF for 3 mg/kg indomethacin to be about 50-200 ⁇ g/kg host weight (50-200 ⁇ g/m2 for humans).
- the effects of ibuprofen in combination with TNF were approximately equal to those of aspirin In combination with TNF.
- the results confirm the best dose of TNF for 30 mg/kg host aspirin.
- CD rats purchased from Charles River Labs were injected with either 200 or 400 ⁇ g/kg of the TNF mutein described above in Example 1 intravenously once at 0 hours.
- CD rats were injected intravenously 24 hours and one hour prior to TNF treatment iv at 0 hours with N-acetyl cysteine (Sigma) at 250 and 100 mg/kg host.
- the data shown in Table VIII are the deaths within 24 hours/total rats treated. It can be seen that the administration of the N-acetyl cysteine reduces the toxicity of the TNF at all doses tested.
- lymphokines or cytotoxins other than TNF may play a role in IL-2 mediated tumoricidal activity.
- IL-2 mediated tumoricidal activity.
- the following experiment illustrates that IL-2 may be as effective as TNF.
- mice Female Balb/c mice were implanted subcutaneously with Meth-A tumor cells as described in Example 1.
- An IL-2 mutein designated des-ala1-IL2- ser125 (with no alanine at position 1 of the mature IL-2 molecule and a serine at position 125 of the mature IL-2 molecule), was prepared by the method described in U.S. Patent No. 4,518,584, supra, isolated from refractile bodies in accordance with the procedure described in EP-A-0 206 828, published December 30, 1986, and formulated as described in U.S. Patent No. 4,604,377 in sodium dodecyl sulfate.
- the designation des-ala1-IL-2 ser125 indicates that the initial alanine of the mature native IL-2 sequence is missing and that the cysteine residue at position 125 of the mature native IL-2 sequence has been substituted with a serine.
- Table IX indicates the results obtained when the IL-2 mutein described herein alone, uric acid alone, and various combination injections of the IL-2 mutein and uric acid were administered intravenously to five mice per group (2-3 repeats) beginning seven days after tumor implantation and once daily for five days, with the uric acid being administered immediately prior to (about 10 minutes) the IL-2 administration when both were administered. Final measurements were made on day 14. The excipient control was injected with PBS. The uric acid doses were all 25 mg/kg of mice weight. The headings are defined in the footnotes of Table I.
- mice resulted in a decrease in toxicity over the IL-2 control; however, the IL-2 was about 2-fold less efficacious when given with uric acid. This decrease is similar in magnitude to what was observed with lower TNF doses and uric acid. It is predicted that longer treatments and an increase in the dose of IL-2 will result in cures of the tumor.
- the preferred biological modifiers for human use are aspirin, vitamin C, vitamin E, ibuprofen, indomethacin, and N-acetyl cysteine.
- IL-2 is typically administered at a level of 3 x 106 units/m2 per day.
- the present invention achieves the following goals: First, the ability of the host or tumor to scavenge radicals is reduced by the combination therapy, for example with BSO, thereby increasing the therapeutic index of the tumor over the host. Second, the biological damage at the host level caused by free radical production is blocked, e.g., by the plasma-mediated radical scavenger, uric acid. Third, the metabolism of the arachidonate cascade, which is influenced by and itself produces reactive radical species in lipid and aqueous compartments, can be modulated to increase the therapeutic index of the lymphokine or cytotoxin. Effective amounts of the biological modifier can be determined for humans based on translations from mice data when human clinical trials are undertaken.
- the present invention is seen to provide a combination of lymphokine or cytotoxin and biological modifier that results in an enhanced therapeutic index relative to the lymphokine or cytotoxin alone in a mammalian host.
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Claims (10)
- Procédé pour la préparation d'une composition appropriée à l'administration à des hôtes mammifères pour le traitement thérapeutique ou prophylactique d'une lésion biologique de l'hôte provoquée par la formation de radicaux libres, qui comprend le mélange, en des quantités pharmacologiquement actives, d'au moins une lymphokine ou cytotoxine d'une espèce de mammifère et d'au moins un modificateur biologique choisi parmi un épurateur de radicaux libres ou un inhibiteur métabolique.
- Procédé selon la revendication 1, dans lequel la lymphokine ou cytotoxine est l'interleukine-2, l'interféron-β, le facteur nécrosant des tumeurs ou le facteur stimulant la formation de colonies-1.
- Procédé selon la revendication 1 ou 2, dans lequel la lymphokine ou la cytotoxine est le facteur nécrosant des tumeurs-α.
- Procédé selon l'une quelconque des revendications 1 à 3, dans lequel le modificateur biologique est choisi parmi l'acide urique, la buthionine sulfoximine, la vitamine E, la vitamine C, la N-acétylcystéine, un rétinoïde, le glutathion, le métronidazole, l'aspirine, l'indométhacine, l'ibuprofène, l'acide nordihydroguaiarétique, l'acide cis-8,11,14-eicosatriène-5-ynoïque, les prostaglandines synthétiques, les leucotriènes synthétiques et les combinaisons d'un ou plusieurs de ces modificateurs.
- Procédé selon l'une quelconque des revendications 1, 2 ou 4, dans lequel la lymphokine est l'interleukine-2.
- Procédé selon la revendication 5, dans lequel le modificateur biologique est l'acide urique et l'interleukine-2 est une désalanyl₁ mutéine avec un reste de sérine dans la position 125 de la molécule de l'interleukine-2 native.
- Utilisation de quantités pharmacologiquement efficaces d'au moins une lymphokine ou d'une cytotoxine d'une espèce de mammifère et d'au moins un modificateur biologique choisi parmi un épurateur de radicaux libres ou un inhibiteur métabolique, pour la production d'une composition pharmaceutique pour le traitement thérapeutique ou prophylactique d'une lésion biologique d'un hôte mammifère provoquée par la formation de radicaux libres.
- Utilisation d'au moins un modificateur biologique choisi parmi un épurateur de radicaux libres ou un inhibiteur métabolique, pour la production d'une composition pharmaceutique pour accroître l'index thérapeutique d'au moins une lymphokine ou une cytotoxine d'une espèce de mammifère dans le traitement thérapeutique ou prophylactique d'une lésion biologique d'un hôte mammifère provoquée par la formation de radicaux libres.
- Utilisation selon la revendication 7 ou 8, dans laquelle la lymphokine ou la cytotoxine est choisie parmi une interleukine, un interféron, un facteur nécrosant des tumeurs ou un facteur stimulant la formation de colonies.
- Utilisation selon l'une quelconque des revendications 7 à 9, dans laquelle la lésion biologique est un cancer, une infection, une lésion provoquée par une tension élevée d'oxygène, une radiothérapie ou une chimiothérapie.
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AT87117149T ATE78700T1 (de) | 1986-11-21 | 1987-11-20 | Therapeutisches gemisch aus einem faenger von freien radikalen oder einem stoffwechsel-hemmer und einem biologisch aktiven protein. |
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US93347586A | 1986-11-21 | 1986-11-21 | |
US933475 | 1986-11-21 | ||
US11364387A | 1987-10-26 | 1987-10-26 | |
US113643 | 1987-10-26 |
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EP0269017A2 EP0269017A2 (fr) | 1988-06-01 |
EP0269017A3 EP0269017A3 (en) | 1989-06-21 |
EP0269017B1 true EP0269017B1 (fr) | 1992-07-29 |
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EP87117149A Expired - Lifetime EP0269017B1 (fr) | 1986-11-21 | 1987-11-20 | Association thérapeutique d'un épurateur de radicaux libres ou d'un inhibiteur métabolique et d'une protéine active biologiquement |
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EP (1) | EP0269017B1 (fr) |
CA (1) | CA1321349C (fr) |
DE (1) | DE3780770T2 (fr) |
ES (1) | ES2042529T4 (fr) |
GR (1) | GR3005314T3 (fr) |
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US5296500A (en) * | 1991-08-30 | 1994-03-22 | The Procter & Gamble Company | Use of N-acetyl-cysteine and derivatives for regulating skin wrinkles and/or skin atrophy |
US5523077A (en) * | 1992-02-06 | 1996-06-04 | Yale University | Composition and method for whitening skin |
GB9226729D0 (en) * | 1992-12-22 | 1993-02-17 | Wellcome Found | Therapeutic combination |
AU673518B2 (en) * | 1993-03-10 | 1996-11-14 | Yale University | Composition and method for Treating skin |
US5686436A (en) * | 1993-05-13 | 1997-11-11 | Hiv Diagnostics, Inc. | Multi-faceted method to repress reproduction of latent viruses in humans and animals |
EP0750496A1 (fr) * | 1994-03-14 | 1997-01-02 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Utilisation d'inhibiteurs de la lipoxygenase en tant qu'agents therapeutiques et d'intervention anticancereux |
US5451405A (en) * | 1994-04-25 | 1995-09-19 | Chesebrough-Pond's Usa Co. | Skin treatment composition |
ES2977792T3 (es) | 2017-09-07 | 2024-08-30 | Childrens Hospital Philadelphia | Composiciones y métodos para el tratamiento de la angiopatía amiloide por cistatina C hereditaria (HCCAA) y otros trastornos neurodegenerativos asociados con depósitos de amiloide aberrantes |
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GB1526205A (en) * | 1974-11-07 | 1978-09-27 | Searle & Co | Treatment of interferon-like material |
EP0177342A3 (fr) * | 1984-10-04 | 1987-12-02 | Genentech, Inc. | Formulations orales de protéines thérapeutiques |
-
1987
- 1987-11-12 CA CA000551719A patent/CA1321349C/fr not_active Expired - Fee Related
- 1987-11-20 ES ES87117149T patent/ES2042529T4/es not_active Expired - Lifetime
- 1987-11-20 DE DE8787117149T patent/DE3780770T2/de not_active Expired - Fee Related
- 1987-11-20 EP EP87117149A patent/EP0269017B1/fr not_active Expired - Lifetime
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1992
- 1992-07-30 GR GR920400697T patent/GR3005314T3/el unknown
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Publication number | Publication date |
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GR3005314T3 (fr) | 1993-05-24 |
ES2042529T4 (es) | 1996-07-16 |
CA1321349C (fr) | 1993-08-17 |
DE3780770T2 (de) | 1993-02-25 |
DE3780770D1 (de) | 1992-09-03 |
ES2042529T3 (es) | 1993-12-16 |
JPS63190831A (ja) | 1988-08-08 |
EP0269017A3 (en) | 1989-06-21 |
EP0269017A2 (fr) | 1988-06-01 |
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